玉米转录因子基因ZmPTF1在转基因烟草中的表达

为了探讨转录因子基因ZmPTF1在玉米种子萌发中的作用,本研究根据GenBank中收录的玉米PTF1序列设计PCR引物,利用普通玉米自交系A318克隆得到ZmPTF1的全长cDNA序列.该序列开放阅读框包括1 446bp碱基,由481个氨基酸组成.本文构建植物表达载体PCAMBIA3301-ZmPTF1转化烟草W38,经过抗生素筛选和PCR鉴定得到转基因阳性烟草,对2株生长正常的T1转基因烟草进行southern杂交分析及RT-PCR检测,结果表明外源ZmPTF1已经整合进了烟草基因组中并且能正常转录并遗传.与对照相比,转基因烟草种子的发芽率、发芽势及萌发种子α-淀粉酶活性显着增高,表明ZmPTF1基因过表达会促进烟草种子提前解除休眠进入发芽阶段,提高烟草种子发芽势和发芽率.该研究结果为进一步研究ZmPTF1基因的功能提供理论依据.     

黄瓜生长素受体同源基因CsAFB和CsTIR在拟南芥中的转化与功能表达

植物生长素受体是生长素信号通路中的重要因子.基于前期克隆得到了黄瓜(Cucumis sativus)生长素受体同源基因生长素信号F-box蛋白基因(auxin signaling F box protein,CsAFB)和转运抑制响应基因(transport inhibitor response,CsTIR),为进一步证实和研究这2个基因的功能,本研究利用拟南芥(Arabidopsis thaliana)Col-0野生型和生长素受体编码基因功能缺陷突变体tirl-1为材料,导入黄瓜生长素受体同源基因,获取纯合转基因系.检测发现,拟南芥突变体tirl-1转入CsAFB/TIR基因后根系发育、外源生长素敏感性和细胞伸长反应均恢复至野生型水平.检测发现,野生型拟南芥中过量表达黄瓜CsAFB/TIR,尤其是Cs TIR基因,植株主根伸长明显受抑,侧根数量剧增,子叶下卷,叶柄上翘,真叶叶缘向离轴面弯曲,顶端优势明显.本研究表明,CsAFB/TIR基因功能类似拟南芥TIR1基因,为黄瓜生长素受体同源基因;过量表达该类基因通过增加生长素受体数量、扩大生长素信号的方式参与调控植物生长发育;为进一步在黄瓜中研究生长素受体功能及作用机理提供理论依据.     

美国豆芋花总皂苷制备及其α-葡萄糖苷酶抑制活性

为高效利用美国豆芋花皂苷类化合物,以皂苷得率和纯度为指标,研究美国豆芋花总皂苷的提取工艺及大孔树脂纯化工艺,并对制备的美国豆芋花总皂苷的抗氧化及α-葡萄糖苷酶活性抑制能力进行研究。结果表明,美国豆芋花总皂苷的提取工艺为提取溶剂90%乙醇,料液比1∶20,温度70℃,提取时间2 h。得到的提取物石油醚脱脂后用水饱和正丁醇萃取,D-101大孔树脂吸附柱纯化的工艺为上样皂苷质量浓度1 mg·m L~(-1),上样量40 m L·g~(-1)树脂,洗脱剂为80%乙醇(pH值6),洗脱剂用量4 BV(树脂柱体积)。制备的美国豆芋花总皂苷样得率4.24%,纯度达65.35%,比粗提物(纯度15.27%)提高了约3倍;抗氧化性能(清除DPPH和ABTS自由基IC50值分别为51.08、29.24μg·m L~(-1))和α-葡萄糖苷酶活性抑制能力(IC50值为83.62μg·m L~(-1))也均优于粗提物(清除DPPH、ABTS自由基IC50值分别为286.51、69.92μg·m L~(-1),α-葡萄糖苷酶的抑制浓度(IC50)值为1 043.57μg·m L~(-1)),表明该工艺可用于高活性美国豆芋花总皂苷的富集和纯化。此外,美国豆芋花总皂苷抑制α-葡萄糖苷酶活性的抑制类型为竞争型抑制,抑制常数为49.68μg·m L~(-1)。本研究结果为开发具有降血糖功能的医药中间体或功能性食品提供了新资源和实践基础。     

甘草总黄酮对金黄色葡萄球菌的作用研究

为了研究甘草总黄酮对金葡菌的抗菌活性以及亚抑菌浓度的甘草总黄酮对金黄色葡萄球菌α-溶血素分泌的影响。首先,采用微量肉汤稀释法测定了甘草总黄酮对6株金葡菌标准菌株的最小抑菌浓度;其次,采用溶血试验、蛋白免疫印迹试验以及荧光定量PCR试验分别在表型功能、蛋白表达和基因转录3个层次确证了其对α-溶血素表达的影响。结果表明：甘草总黄酮具有良好的抗金葡菌作用,其最小抑菌浓度范围为4~16 μg/mL,而亚抑菌浓度的甘草总黄酮能浓度依赖性地降低金葡菌α-溶血素的分泌。     

Use of a cyclic high-pressure sodium lamp to inhibit flowering of chrysanthemum and velvet sage

Photoperiod is commonly controlled in the commercial production of ornamental crops to induce or prevent flowering. Flower induction in short-day (SD) plants can be prevented or delayed when the natural daylength is short by providing low-intensity lighting during the dark period. A stationary high-pressure sodium (HPS) lamp with an oscillating aluminum parabolic reflector (cyclic HPS) has been developed to provide intermittent lighting to greenhouse crops. We determined the efficacy of a cyclic HPS lamp at preventing flowering in SD plants garden chrysanthemum [Chrysanthemum × grandiflorum (Ramat.) Kitam.] ‘Bianca’, pot chrysanthemum ‘Auburn’, and velvet sage (Salvia leucantha L.) relative to traditional night interruption (NI) lighting strategies. Plants were grown in a glass-glazed greenhouse at a mean daily temperature of 19.5–20.7 °C with natural SD photoperiods. NI lighting was delivered during the middle of the night (2230–0230 h) from a 600 W cyclic HPS lamp mounted at one gable end of the greenhouse or from incandescent (INC) lamps that were illuminated for the entire 4 h (CONT INC) or for 6 min every 30 min for 4 h. Plants under cyclic HPS were grown at lateral distances of 1, 4, 7, 10, or 13 m from under the lamp. Control plants were grown under an uninterrupted 15 h skotoperiod. As the distance from the cyclic HPS lamp increased from 1 to 13 m, the maximum irradiance measured during the NI decreased from 25.4 to 0.3 μmol m⁻² s⁻¹ and time to visible inflorescence (VI) and the number of nodes at VI decreased. All species had a VI within 54 d, but ≤10% of plants flowered when grown at a lateral distance of 1 or 4 m from the cyclic HPS lamp or under CONT INC. Plants grown without NI had a VI 2 to 15 d earlier and flowered 7 to 24 d earlier than plants grown at 10 or 13 m from the cyclic HPS. All garden chrysanthemums flowered under cyclic INC, whereas velvet sage and pot chrysanthemum had 15% and 35% flowering, respectively. These results indicate that a cyclic HPS lamp can be used effectively to delay flower induction and prevent flowering in these species when NI is delivered at ≥2.4 μmol m⁻² s⁻¹.     

甘蓝自交不亲和决定因子的体外表达和相互作用的检测

 S位点受体激酶(SRK)和S位点富含半胱氨酸蛋白/S位点蛋白11(SCR/SP11)分别为甘蓝自交不亲和(self-incompatibility, SI)信号传导的雌雄决定因子。为了深入研究两者的作用机理和进行人工调控,本研究以结球甘蓝ZQ为材料,利用pET·NusA融合蛋白表达系统,将包含SRK胞外域和跨膜域的mSRK蛋白和SCR蛋白在大肠杆菌BL21中融合表达,经SDS-PAGE电泳检测表达出融合蛋白大小分别约为116 kD和74 kD。进一步将两融合蛋白进行体外相互作用的检测,结果表明mSRK与SCR蛋白能够相互结合形成稳定的复合体,这为下一步实现SRK-SCR复合体聚合与解离的人为调控提供了技术平台。     

酸性α-醋酸萘酯酶染色法的改良

通过对酸性α-醋酸萘酯酶染色过程中的几个重要因素进行研究,确定了其最佳处理条件,并探讨了几个因素间的相互作用。结果表明,固定液用2.5%戊二醛的效果要优于甲醛-丙酮混合液;用pH值5.8～6.2的孵育液孵育1.5h,甲基绿复染1～10s效果最佳。变化各因素的处理条件对最终的观测效果和结果都有影响,其中以甲基绿复染时间过长影响最大。同时,该染色法应用于贵妃鸡的T淋巴细胞计数中,4次重复的结果表明,贵妃鸡酸性α-醋酸萘酯酶阳性细胞率为37.86±1.89%,各重复间差异不显著。     

温度、丙烯和1-MCP对西洋梨果实乙烯合成和乙烯受体ETR1同源基因表达的影响

研究了贮藏温度、丙烯和 1 MCP (1-甲基环丙烯 )处理对帕斯—卡桑 (Passe -Crassane)西洋梨果实成熟的影响。结果表明 :低温可促进果实的乙烯合成 ,未经低温处理的果实 ,用丙烯处理 ,不能正常合成乙烯。丙烯促进乙烯合成的效应随冷藏时间延长而增强。1 MCP处理可抑制冷藏果实的乙烯合成。定量PCR分析结果表明 ,果实中乙烯受体ETR1同源基因的表达不受低温的调节 ;但冷藏后升温或升温后用丙烯处理时 ,mRNA含量降低     

Protective effect of C1q/tumor necrosis factor-related protein 3 on vascular endothelium in rats with hyperuricemia

AIM To study whether C1q/tumor necrosis factor （TNF）-related protein 3 （CTRP3）protect vascular endothelium in rats with hyperuricemia and its potential mechanisms. METHODS An animal model of hyperuricemia was established by using male SD rats drinking 10% fructose water （n=10）. The rats drinking normal water served as normal controls （n=10）. After 12 weeks, the rats were given a single injection with Ad-CTRP3 or Ad-GFP. The experiment was ended at 14th day after transfection.The serum levels of uric acid and nitric oxide （NO） were evaluated. The serum contents of TNF-α and interleukin-6 （IL-6） were measured by ELISA. HE staining and TUNEL assay were used to assess the morphological changes of intima and apoptosis of endothelial cells in thoracic aorta, respectively. The mRNA levels of endothelial nitric oxide synthase （eNOS）, TNF-α and IL-6 were detected by RT-qPCR. The protein levels of CTRP3 and Toll-like receptor 4 （TLR4） were determined by Western blot. RESULTS Compared with normal control group, the rats with hyperuricemia showed lower CTRP3 and higher TLR4 protein levels in the thoracic aorta （P<0.05）. Hyperuricemic rats had higher serum contents of uric acid, TNF-α and IL-6 （P<0.05）. Also, the intima structure disturbance of thoracic aorta, increased apoptotic rate, higher mRNA levels of TNF-α and IL-6 as well as lower mRNA levels of eNOS were observed （P<0.05）. By contrast, CTRP3 over-expression decreased TLR4 protein levels, reduced inflammatory cytokines, and obviously improved the morphology and function of thoracic aorta in the rats with hyperuricemia. CONCLUSION CTRP3 protect vascular endothelium in rats with hyperuricemia maybe via down-regulation of TLR4- mediated inflammatory signaling pathway.     

CYP450 epoxygenase/EET pathway attenuates adipose inflammation of obese mice through HIF-1α

AIM To investigate the effects of cytochrome P450 （CYP450） epoxygenase/epoxyeicosatrienoic acid （EET） pathway on insulin resistance in obese mice, and to explore the possible mechanisms. METHODS High-fat diet-induced obesity model was established in C57BL/6Cnc mice, and the obese mice were randomly divided into 3 groups, including obesity group （treated with saline; n=10）, EET group （treated with 11,12-EET; n=10） and EET inhibitor 14,15-epoxyeicosa-5（Z）-enoic acid （EEZE） group （n=10）. Normal C57BL/6Cnc mice （n=10） treated with saline served as control. Protein expression of CYP2J2 （one of CYP450 epoxygenases） and hypoxia-inducible factor-1α （HIF-1α） was measured by Western blot. Vessel-like structure was detected by immunofluorescence staining. The serum levels of insulin, tumor necrosis factor-α （TNF-α）, interleukin （IL）-1β, IL-6 and monocyte chemoattractant protein-1 （MCP-1） were measured by ELISA. RESULTS In obese mice, homeostasis model assessment of insulin resistance （HOMA-IR） values were increased, the protein level of CYP2J2 was reduced, and the protein level of HIF-1α was increased in adipose tissues as compared with the controls （P<0.05）. The serum levels of MCP-1, IL-1β, IL-6 and TNF-α were also significantly increased in obese mice （P<0.05）. After treatment with 11, 12-EET, the HOMA-IR values were decreased compared with vehicle-treated obese mice, HIF-1α expression levels were decreased in the adipose tissue, and the serum levels of MCP-1, IL-1β, IL-6 and TNF-α were reduced （P<0.05）. Immunohistochemical results of adipose tissue from vehicle-treated obese mice showed a marked decrease in vessel-like structures （CD31-positive） compared with normal control mice （P<0.05）. EET treatment significantly increased the newly formed vessel-like structures in the visceral adipose tissues of obese mice as compared with vehicle-treated obese mice （P<0.05）. CONCLUSION High-fat diet-induced obesity and insulin resistance are closely related to the CYP450 pathway. Exogenous EETs effectively decrease obesity-induced insulin resistance possibly through pro-angiogenesis and attenuation of hypoxia and inflammation.